PROJECT SUMMARY During an infection, the bacterial envelope mediates interactions between bacteria and their hosts through structures such as adhesins and polysaccharides. The structure and composition of the cell envelope are also crucial determinants of whether a bacterium will be defeated by or resistant to antibiotics. For example, Gram- negative bacteria are naturally resistant to many antibiotics and detergents because their outermost envelope layer, the outer membrane, excludes hydrophobic small molecules that otherwise can cross typical lipid bilayers. Therefore, understanding how Gram-negative bacteria build their cell envelope and maintain envelope integrity and impermeability is crucial for the development of new antibacterial strategies to combat infections. Our long-term goal is to understand at the molecular level how Gram-negative bacteria build their cell envelope. The Gram-negative envelope is delimited by two lipid bilayers (the inner and outer membranes), which are separated by an aqueous compartment (the periplasm) where a thin layer of peptidoglycan cell wall resides. Although many factors involved in the biogenesis and maintenance of the cell envelope have been identified in the last few decades, there are still many envelope proteins whose function remains unknown. This proposal seeks to investigate the AsmA-like protein clan, which includes proteins that have been reported to be important for bacterial-host interactions, as well as for maintaining the impermeability to antibiotics and the integrity of the cell envelope of some Gram-negative bacteria. Furthermore, we have found that members of the AsmA-like protein clan are ubiquitously present in diderm bacteria, even Gram-negative endosymbionts that have undergone massive genome reduction. From these lines of evidence, we hypothesize that AsmA-like proteins perform a yet-to-be-identified fundamental function in the envelope of double membrane bacteria. We also argue that this function has remained elusive in organisms used as models to study envelope biogenesis and function because of redundancy among AsmA-like paralogs. Indeed, our preliminary data has uncovered functional redundancy among AsmA-like paralogs in the Gram-negative bacterium Escherichia coli. Here, we propose to take advantage of the genetics system, and the extensive base of knowledge and experimental tools available to study the cell envelope of E. coli in order to conduct the first in-depth functional characterization of multiple AsmA-like paralogs. We expect that the proposed studies will identify: 1) the contribution of each AsmA-like paralog to envelope homeostasis; 2) the functional relationship between AsmA- like paralogs; 3) which pathways are affected by the loss of AsmA-like proteins; and 4) potential functional partners of AsmA-like proteins. To achieve these goals, we will combine genetic and biochemical approaches together with detailed phenotypic characterization, a combination that has been proven effective for the identification and characterization of envelope biogenesis factors. The knowledge gained from the proposed studies will contribute to the development of novel therapies to combat infections by Gram-negative pathogens.